DESCRIPTION: This application proposes further research on microbial secondary metabolism and polyketide biochemistry. We have pursued this work for several years through a study of tetracenomycin (tcm) C production in Streptomyces glaucescens. The carbon skeleton of this metabolite is made by a type II polyketide synthase (PKS) consisting of at least five proteins, produced by the tcmJKLMN genes, with the possible involvement of the fabD gene. A major goal of our research is to elucidate the properties of each component enzyme of the type II Tcm PKS through studies of the purified enzymes in vitro. In this way will we be able to determine the factors controlling poly-beta-ketone synthesis and cyclization, without the uncertainties of whole cell biotransformations. We also will establish the properties of new combinations of existing PKS genes or novel ones constructed from domains of type I and type II PKS genes. Although non-functional enzymes or enzyme complexes will occasionally be produced, without always being able to understand the reason for a negative result, such research should be fruitful often enough to justify it. This work should also teach us important concepts about the PKS mechanisms and, of equal importance, provide natural products that can be evaluated for their biological activity. Consequently, during the next five years we propose to extend our work on microbial PKSs in the following two main areas, with the goals specified. (1) BACTERIAL TYPE II PKSs FOR THE BIOSYNTHESIS OF POLYCYCLIC AROMATIC COMPOUNDS. For the tetracenomycin biosynthesis: (a) Purify the TcmJ, TcmK, TcmL and FabD enzymes, (b) reconstitute the Tcm PKS in vitro and (c) obtain solid state 3-D structures of TcmI and TcmN by x-ray crystallography. For daunorubicin biosynthesis in Streptomyces peucetius.: (a) Express and purify the DpsC, DpsD, DpsE and DpsF enzymes, (b) determine if the DpsC and DpsD enzymes alone govern the starter unit specificity and (c) obtain solid state 3-D structure of DnrD by x-ray crystallography. For jadomycin biosynthesis in Streptomyces venezuelae: (a) Express and purify the Jad-Orf4 polyketide cyclase. (2) HYBRID AROMATIC POLYKETIDES MADE WITH BACTERIAL AND/OR FUNGAL GENES. (a) Analyze the mechanism of polyketide cyclases by studying the properties of hybrid sets of PKS genes containing the tcmJ, tcmN, dpsF or jad-ORF4 genes in different combinations. (b) Analyze the mechanism of fungal polyketide synthases and cyclases by studying the properties of hybrid sets of PKS genes containing combinations of the 6MSAS, PKSst and genes. (c) Analyze hybrid type II PKSs made from gene cloned from soil DNA.